Chapter 7

Question 1

Define TSG and oncogene.
Include if muations are dominant or recessive and if mutations should be loss or gain of function mutations to activate them.

Answer 1

Oncogene: Gene whose protein product contributes to the development or progression of cancer. The normal gene is the proto-oncogene. This must be activated, gain of function. The mutation is dominant in that only 1 allele must be mutated to promote the malignant phenotype.

TSG: LOF mutation. It is recessive and requires BOTH alleles to be mutated to promote malignancy. More likely to cause inherited cancer.

Question 2

4 methods of oncogene activation include (describe how each causes cancer progression):

Retroviral Transduction

Translocation

Gene Amplification

Proviral Insertion

Answer 2

Retroviral Transduction -
ex: insertion of v-myc (viral myc) as opposed to c-myc (cellular myc)
The viral version of the normal cellular protein usually has a number of mutations that promote cancer such as constitutive activation of the protein, decreased normal negative regulation on protein function, and the presence of a strong viral promoter that leads to increased gene expression. USUALLY W/ ACUTE TRANSFORMING RETROVIRUSES

Translocation -
ex: Bcr-abl in Philadelphia chromosome in CML where the fusion protein has stronger tyrosine kinase activity.
If 1 gene is placed in front of another gene w/ a more active promoter leading to increased expression. Other fusion proteins such as c-myc in front of IG locus - leads to c-myc expression.

Gene Amplification -
Multiple copies of the gene leading to increased expression. Can be homologously staining regions (w/in chrom) or double minute chromosomes (outside of chrom)

Proviral Insertsion -
CHRONICALLY TRANSFORMING RETROVIRUSES. Require long latency period before seeing transformation. DN add genetic material to cell but rather can cause a mutation based on where they inserted.
Ex: If they inserted the retrovirus near a protooncogene, that cell may have a growth advantage. Strong retroviral promoters can cause overexpression of the protooncogene and lead to tumor formation.
Ex: Avian leucosis virus inserts near c-myc leading to bursal lymphoma.

Question 3

Oncogene products can be classified as either:
growth factors
growth factor receptors
Ras oncoproteins
Cytoplasmic protein kinases
Transcription factors
Anti-apoptotic proteins

Categorize the following and where appropriate, name the function of the protein/gene:

v-sis, fos, Her2/Neu, VEGFR, H-ras, BCR-ABL
Met, PDGFR, Myc, Kit, Bcl-2, EGFR, Src, Jun

Answer 3

v-sis - GF - PDGF
fos - TF 
Her2/Neu - GFR - heregulin receptor
VEGFR - GFR
H-ras - oncoprotein - GTPase
BCR-ABL - protein TYROSINE kinase
Met - hepatic GFR
PDGFR - platelet derived GFR
Myc - TF
Kit - GFR - stem cell factor
Bcl-2 - anti-apoptotic protein
EGFR - epidermal GFR
Src - TYROSINE kinase
Jun - TF

Question 4

The multi-hit hypothesis states that cooperation bt multiple mutations in oncogenes and TSG are needed for transformation to proceed. List the changes a cell must undergo to become cancerous (5 steps):

Answer 4

1. proliferate independently of growth signals
2. circumvent programmed cell death
3. replicate indefinitely
4. induce vascular formation
5. invade tissues
   ON EXAM!

Question 5

Explain how receptor tyrosine kinases are activated and promote cell growth and list at least 3 ways that can lead to RTK activation (which then promotes cancer development)

Answer 5

Growth factors are the ligands for RTKs
RTKs are cytoplasmic bound receptors.
After they bind their ligand, they can activate the others intracellular domain via phosphorylation of cytoplasmic signaling molecules (such as GTPases).
In cancer, RTKs can be constitutively activated leading to excess GF stimulation and cell growth.

Examples of activation:

1. ligand overexpression
2. overexpression of RTK leading to increased dimerization and t/f activation
3. mutation in the binding pocket leading to constitutive activation of RTK w/o ligand (as w/ c-KIT in some MCT!)

Question 6

Give an example of a Mab specifically targeted against RTKs

Answer 6

Herceptin (trastuzumab) - HER2-NEU

Question 7

Describe the function of the ras protein family

Answer 7

They are genes which encode for G coupled proteins which phosphorylate effector molecules in response to signaling cues in the cell.
Inactive when bound to GDP.
Active when bound to GTP - can give Ph to another molecule.

Question 8

How is RAS targeted for anti-cancer therapy? To answer this, think of and explain how RAS normally functions and how we can interfere w/ it…

Answer 8

RAS activation (to GTP form) and localization to cell membrane are essential for normal function. Farnesyltransferase (FTase) is the enzyme that adds a farnesyl lipid moiety to H-Ras. FTase inhibitors can prevent the RAS localization to the cell surface by inhibiting this reaction….however, this does not work for K-ras (more commonly mutated in people) bc K-ras uses geranylgeranyltransferase (GGTIs then needed)

Question 9

Raf-1 and MEK are what type of oncogenes (what is their normal function?)

Answer 9

They are cytoplasmic kinases that are downstream of RAS and are nb in cell signaling for growth and survival.

(ALSO review ERK functions…should be added to major diagram)

Question 10

BCR-ABL is inhibited by what specific drug?
Describe how the drug works in this case. Also list the other proteins in the same oncogene function/class that are responsive to treatment by the same drug.

Answer 10

Imatinib mesylate (gleevac) targets the ATP-binding region of the BCR-ABL tyrosine kinase.

c-KIT and PDGFR

Question 11

Why are transcription factors less favorable as cancer therapy targets?

Answer 11

Located in the nucleus - makes them more difficult to access

Question 12

Apoptosis is generally characterized by the release of cytochrome C from the ______ to the ______ which triggers apoptosis by the activation of what enzymes? Cytochrome c release is regulated by what apoptosis protein?

Answer 12

Mitochondria
Cytoplasm
caspases
BCL-2

Question 13

Are these pro or anti-apoptosis?
Bcl-2
Bcl-xl
Mcl-1
Bax
Bid

Answer 13

Bcl-2, Bcl-xl, Mcl-1 - all anti (pro-survival)

Bax, Bid - pro-apoptotic

Question 14

Describe the Knudsen 2 hit hypothesis

Answer 14

Recessive mutations in TSG required that both copies of the alleles be affected prior to tumor development. Results in early formation if 1 mutation is inherited or later in life if 2 random mutations occur

Question 15

TSG are divided into gatekeepers and caretakers. Describe the functions of each group and name 2 examples of each.

Answer 15

Gatekeepers:
Directly affect tumor growth by counteracting proliferation or promoting cell death.
ex: RB, p53, PTEN

Caretakers:
Normally act to provide genetic stability so with mutations there is genetic instability, increased mutation rate, promotion of oncogenes, and inactivation of gatekeepers
ex: ATM nb in radiosensitivity, XP genes nb for NER, and other DNA repair enzymes.

Question 16

What is Li Fraumeni syndrome characterized by?

Answer 16

Heterozygous mutation in p53
Rare autosomal dominant disorder
Leads to many tumors in SAs and CAs

Question 17

Name 3 changes in the cell that can lead to p53 up regulation:

Describe how p53 can prevent G1-S cell cycle progression:

Answer 17

1. Hypoxia, DNA damage, oncogene activation
2. In response to DNA damage, p53 upregulates p21 WAF. p53 is a transcription factor for p21 WAF1. P21 is a cdki preventing G1-S transition and G1 arrest preventing the cell with damaged DNA from entering synthesis phase. P53 can also induce apoptosis in cells w/ RT or chemo damage

Question 18

What is the function of Mdm-2?

Answer 18

Antagonizes p53 transcriptional activity, promotes export of p53 from nucleus and targets it for UB degradation

p53 activates mdm-2 transcription to act as negative feedback on itself in normal cells

Question 19

PTEN - what is its normal function and what is seen in the cell when PTEN is lost?

Answer 19

1. PTEN dephosphorylates PIP3 (made by PI3K) and inhibits PI3K. PI3K is nb for signaling, regulating proliferation, survival, adhesion, metabolism of glucose and cytoskeleton organization
2. LOSS of PTEN leads to increased PIP3 and increased AKT activation
3. With abnormal PTEN, you are less sensitive to cellular stressors, have increased threshold for inducing apoptosis
4. Expression of PTEN leads to inhibition of proliferation, migration and cell spreading

Question 20

Retinoblastoma has been found to be mutated in inherited ocular tumors. Explain the role of Rb in regulation of cell cycle progression. Specifically discuss how phosphorylation effects Rb, what proteins regulate Rb phosphorylation and the interaction of Rb with particular transcription factors that cause cell cycle progression or cell cycle arrest

Answer 20

Rb arrests cells in G1 phase, Rb function is dependent on protein phosphorylation!!! Rb is phosphorylated by CDKs.

Rb is INACTIVE when hyperphosphorylated - allows cell proliferation.
ACTIVE when HYPOphosphorylated - inhibits G1 progression to S

Rb regulated by

• Cyclin D/CDK 4 and 6 in early G1
• Cyclin E/CDK 2 in late G1
• Cyclin A/CDK 2 maintains Rb phosphorylation during S phase

Again, phosphorylation by these cyclin/CDK complexes keeps Rb inactive and allows cell cycle progression.

Actions of Rb can be explained by interaction with E2F transcription factors. When Rb is phosphorylated, E2F is free to bind DNA and transcribe genes that participate in DNA replication and cell cycle progression. When Rb is not phosphorylated, it can bind E2F keeping it from binding DNA, preventing transcription of necessary proteins for cell cycle progression.